大頭鰱卵膜主要醣蛋白的構造折究

Abstract

卵膜的功能有保護卵的本身、促成精卵結合和避免多精化( polspermy )出現。對大多數的魚類而言，精卵的結合是經由卵膜上的小孔( micropyle )來結合，而其餘的卵膜角色除了保護魚卵本身外則未知。本篇論文的目的是想探討大頭鰱卵膜主要成份的結構。 我們將大頭鰱的卵膜萃取液用雙向電泳( 2D-gel )分離，割取膠體內BH carp ZP2的膠體，然後用蛋白分解酵素水解（ in gel digestion ) ，經逆相HPLC分離，得到片段的 peptide sequence ，用這些 peptide sequence 和比目魚的 wf♀（ZP2）與鯉魚的 ZP2 比對，找出合適的 primers ，進行三次的PCR ，我們得到 1061 nucleo- tides （相當於354個胺基酸），這 BH carp ZP2 sequence 與哺乳的 zona pellucida 2 系列相似性大約是36%。而大頭鰱的 BH carp ZP2 和比目魚的 wf♀ 與鯉魚的ZP2最大不同在大頭鰱的 BH carp ZP2 沒有 N- 端的 repeat domain ，其餘部份相似性非常高（>70%）。 我們用抗 BH carp ZP2 的血清對魚卵染色，發現大頭鰱卵膜的內膜有存在 BH carp ZP2。

Egg envelope has the function of protecting oocyte itself. It can also promote sperm - egg binding and prevent oocyte from polyspermy. In most fish, sperm - egg binding is achieved via the micropyle of egg envelope. Except oocyte protection, other functions of chorion (egg enveplope of fish ) are unknown. The purpose of this work is try to investigate the major glycoprotein structure of big head carp chorion. We employed 2D-gel technique to separate the big head carp chorion protein and carried out the in-gel enzymatic digestion on a predominant spot tentatively named BH carp ZP2. After RP-HPLC purification of the digests, some peptide fragments were obtained. These peptide sequences were compared with those of winter flouder ZP2 and carp ZP2 in order to search the proper primers for PCR. We performed PCR three times and consequently elucidated in -frame 1061 nucleotides beginning from the N-terminus codon (equivalent to 354 amino acid residues). In comparison with the only known ZP2 of fish, winter flouder ZP2 ,the deduced BH carp ZP2 protein sequence still lacks the C-terminal portion of about 30-40 amino acid residues. So far, they have the homology of 76% as the maximum similarity alignmemt. It is interesting that BH carp ZP2 is devoid of repeat domain existing in the N-terminal domain of winter flounder ZP2 . When compared with ZP2 of Mammalia, BH carp ZP2 showed homology of around 36%. The results of immunocytochemical staining revealed the presence of ZP2 in the innermost membrane of chorion. The presence of ZP2 in the innermost membrane might be related to its presumed role of polyspermy prevention.